?-NGF stimulates steroidogenic enzyme and VEGFA gene expression, and progesterone secretion via ERK 1/2 pathway in primary culture of llama Granulosa cells

The beta-nerve growth factor (?-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ?-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ?-NGF,?-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ?-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3?-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ?-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ?-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ?-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway. © 2021 Elsevier B.V., All rights reserved.

llama, progesterone, cells, granulosa, pathway, steroidogenic, also